Abstract While contemporary research has shown Type 1 diabetes (T1D) is associated with intestinal dysbacteriosis and leakage, it is not known whether these factors cause the disease. Establishing a causative link between the onset and progression of T1D and dysbacteriosis is critical for developing effective prevention strategies. The overarching hypothesis of this application is that T1D is preceded by pancreatic bacterial exposure, which promotes an anti- bacterial response, pancreatic inflammation, insulitis, and autoimmunity. This hypothesis was formulated based on our preliminary data demonstrating: 1) heightened anti-bacterial responses in juvenile T1D; 2) pathologic responses by islets to bacteria overrepresented in the T1D microbiome; 3) pancreatic inflammation and insulitis in a mouse model of experimental leakage of bacteria into the pancreas; and 4) blockade of the anti-bacterial response protects islets from insulitis. In aim 1 we will determine whether human T1D development is preceded by a cellular and humoral anti-bacterial response and establish an association with the duodenal microbiome. We will test whether bacteria-responsive MAIT cells are activated before clinical disease develops and determine if they are differentially activated by overrepresented bacteria. Next, we will discover whether the increased anti-bacterial IgA response in T1D is directed at overrepresented bacteria. We will profile the duodenal microbiome and determine if it is associated with these defense mechanisms. In aim 2 we will ascertain whether pancreatic exposure to bacteria leads to immune activation, insulitis, and hyperglycemia. We will determine whether pancreatic islets show distinct responses to specific T1D-associated bacteria including R. gnavus, B. Dorei, and S. infantarius. Lastly, we will interrogate the mediators of inflammation and insulitis following pancreatic exposure to overrepresented bacterial species and test strategies to block the response. To accomplish this task, we have established a novel mouse animal of intestinal dysbiosis and leakage to test therapeutic interventions. The proposed experiments will identify pathogenic mechanisms that link intestinal dysbiosis and leakage to T1D uncovering new targets for prevention. These targets will undergo preclinical testing in this application. This submission is responsive to the U01 Cooperative Study Group for Autoimmune Disease Prevention (CSGADP) RFA. Broad area of interest: Pathways, mechanisms, and means best suited to practical preventive interventions. Specific research topic: Identification and elucidation of cellular and immune pathways that may provide targets for preventive interventions.